The present invention relates to a device for feeding a chromatography flow coming from a flow source, in particular from a liquid chromatography (LC) separating unit or a peak sampling or trapping unit, in part to at least a first decision detector unit and/or at least a first destination detector unit and in part to at least a second destination detector unit, comprising a first capillary line coming from the flow source, a second capillary line leading to the first decision detector unit and/or to the first destination detector unit, and a third capillary line leading to the second destination detector unit, the first, second and third capillary line being connected with one another by means of a flow splitter splitting the flow coming from the first capillary line into two parts, a first part being fed into the second capillary line and the second part being fed into the third capillary line.
The invention further relates to an apparatus for carrying out coupled liquid chromatography (LC) and at least two spectrometry measurements, comprising a LC separating unit, at least a first decision detector unit and at least a first destination detector unit and at least a second destination detector unit, in which the device mentioned before is used.
An apparatus and a device of the kind mentioned before, which are generally known are used in liquid chromatography (LC), in particular in high performance liquid chromatography (HPLC). Liquid chromatography is a known method for separating components of trace elements within liquid substrates to be analyzed.
Further, it is well-known to combine LC with nuclear magnetic resonance (NMR) spectroscopy measurements. The LC is used to separate components of a sample, which are selected in a LC detector, and the selected components were measured afterwards using NMR as the first destination detector unit. The coupling of LC with NMR is nowadays a well-known and accepted technique in science and industry.
For the technique of LC-NMR, four automation modes are known, as they are xe2x80x9con-flowxe2x80x9d, xe2x80x9cstopped-flowxe2x80x9d, xe2x80x9ctime-slicingxe2x80x9d and xe2x80x9cloop-samplingxe2x80x9d.
In a pure on-line coupling, the NMR detector is directly coupled after the liquid chromatograph. In this on-line mode the separated peaks are fed from the LC continuously into the NMR detector to be spectrometrically examined on-line therein.
As an alternative to the on-line mode the stopped-flow technique is used, wherein the flow pump of the LC is stopped as long as a component is investigated inside the NMR detector.
The peak-sampling mode is a mode in which single separated peaks coming from the LC are selected and intermediately stored in a peak sampling unit for later successive investigation in the NMR detector.
The time-slicing mode is a clocked mode in which the LC peaks can be investigated in equally timed fractions to observe spectroscopic changes over a certain elution period.
Right from the beginning of commercializing the combined method of LC-NMR, the idea to hyphenate LC-NMR with additional sophisticated detection methods like mass spectroscopy (MS) as the second destination detector unit existed. Early attempts to combine LC-NMR with MS in 1996 have shown that the information obtained from such a combination was unsurpassed, in particular the selectivity of the peak selection of the LC peaks for further NMR investigation is increased substantially. As described by John P. Shockcor et al. in Anal.Chem. 1996, 68, 4431-4435, the advantage of combined LC-NMR-MS is that the structural information available from the complementary spectroscopic techniques provides rapid confirmation of the identity of the components of the sample.
Heretofore, in order to connect the LC-NMR system with the MS, in all cases just a flow splitter was hooked into the flow path, i.e. the flow splitter splits the flow coming from the first capillary line coming from the LC into two parts, the first part being fed into the second capillary line and the second part being fed into the third capillary line. The second capillary line leads to the LC detector as the first decision detector unit and/or the NMR detector as the first destination detector unit, while the third capillary line leads to the MS as the second destination detector unit. The split ratio between the first part of flow and the second part of flow varied from case to case between 50:1 and 20:1. The larger part of flow is used to feed the NMR detector, and the lower one for the more sensitive MS detector. In general, the users of the LC-NMR-MS system tried to adjust the timing for the MS such that the time a separated peak needed to reach the NMR was equal to or longer than the time to reach the MS.
Depending on the position of the splitter in the entire LC-NMR-MS system, it was possible to use the MS signal to trigger a stop of the chromatography for an NMR measurement, but not to use the MS signal to trigger the storage of the peak in a loop of the peak-sampling unit. All known systems, however, are restricted in the flexibility of their use. The known systems do not take into account that the time scales of the NMR measurements and the MS measurements are quite different. The NMR measurement runs on a time scale which is longer than the time scale of the MS measurements.
Whereas in the preceding description reference has been made to a coupled LC-NMR-MS system, the present invention is not restricted to such a combination, but can be used for other combinations of LC with at least two destination detectors, like for example infrared detectors or light-scattering detectors.
In the present description, a xe2x80x9cdecision detectorxe2x80x9d is to be understood as a detector, the signals of which are used for subsequent actions of the device or apparatus, while a xe2x80x9cdestination detectorxe2x80x9d is to be understood as a detector for detailed investigation and analysis of a sample peak.
It is therefore an object of the present invention to improve a device and an apparatus of the kind mentioned at the outset which allow a coupling of a flow source, in particular a LC separating unit with at least two detector units with a high degree of flexibility in using different modes of LC.
This object is achieved in terms of a device mentioned at the outset in that a first switchable valve means is provided which is connected to the third capillary line and to the second destination detector unit, which has at least two operating positions, wherein in at least a first group of at least one operating position the third capillary line is connected directly to the second destination detector unit, and wherein in at least one second group of at least one operating position the third capillary line is connected to at least one delay line.
By virtue of the first switchable valve means, the flexibility in use of the device according to the invention is advantageously increased. When the first valve means is in the at least one first operating position, the sample flow can quickly reach the second destination detector unit, for example a MS detector, so that the second destination detector unit signal can be used to trigger one of the LC modes which was not possible heretofore with the known systems. In other words, one of the dominant benefits of the present invention is that the second destination detector can advantageously be used as a decision detector for triggering subsequent actions of the device and/or apparatus. In particular, if the second detector unit comprises a MS detector, the high sensitivity of the MS signal can be used for triggering further action and choice of the LC mode, for example on-flow, stopped-flow or loop-sampling action, in particular for selecting peaks of interests from the sample flow. On the other hand, when the first valve means is in the at least one second operating position, this can be used for the sample flow to reach the second detector unit with a certain delay. This can be advantageously used in the on-flow mode in case that the delay is matched to the path from the splitter to the first detector unit, to simultaneously carry out measurements in the first destination detector unit and in the second destination detector unit and thus to have direct comparison of the on-flow data between the first destination detector unit and the second destination detector unit, e.g. a NMR detector and a MS detector. In the stopped-flow mode, the delay line can be used to intermediately park a sample peak therein for later investigation in the second destination detector unit. The flexibility of the device according to the invention, therefore, is highly enhanced.
In a preferred embodiment, the first decision detector unit comprises a chromatography (LC) detector, and the path from the Ad splitter to the second destination detector in the at least one first operating position of the first valve means is matched to the path from the splitter to the chromatography (LC) detector.
With this feature the first part of the sample flow and the second part of the sample flow reach the chromatography detector and the second destination detector unit substantially at the same time, wherein a small time difference in the range of a few seconds is not critical and can be compensated by a suited software. Both information coming from the chromatography detector and from the second destination detector unit can be advantageously combined to make decisions for further actions of the system. The advantage of this double information is an enhanced selectivity of the LC peak selection.
In a further preferred embodiment, the first destination detector unit comprises a spectrometry detector in which a measurement runs on a long-time scale compared to the second destination detector unit in the at least one second operating position of the first valve means, the third capillary line is connected to the second destination detector unit, and the path from the splitter to the second destination detector in the second operating position of the first valve means is matched to the path from the splitter to the first destination detector including the length of the flow cell of the first destination detector.
This feature has the advantage that a simultaneous on-flow measurement can be carried out in the first spectrometry detector, e.g. a NMR detector, and in the second destination detector unit, e.g. a MS detector, further having the advantage to be able to directly compare the on-flow data between the first spectrometric measurement and the second spectrometric measurement.
In a further preferred embodiment, in the at least one second operating position the third capillary line is disconnected from the second detector unit, while a further flow source, in particular a flow injection device, is connected to the second destination detector unit.
With this feature, in particular in case of a stopped-flow mode of the LC, while a measurement is carried out in the first destination detector unit, the second destination detector unit can be advantageously used to carry out further measurements on further sample peaks which are fed into the second destination detector unit from the second flow source. This feature is in particular useful in case that the first destination detector unit needs substantially more time to accumulate data than the second destination detector unit, because during the long-time measurement in the first destination detector unit several other measurements can be carried out in the second destination detector unit.
In a further preferred embodiment, at least one dilutor pump is connectable to the third capillary line for feeding at least one solvent into the third line.
This feature has the advantage that in the stopped-flow mode of the LC during a measurement in the first destination detector unit, one or more peaks parked in the at least one delay line can be fed into the second destination detector unit by means of the dilutor pump and a suited pushing solvent. By means of the dilutor pump, a flow rate for feeding the parked peak into the second destination detector unit can be chosen different from the flow rate generated by the chromatography pump. Furthermore, the sample flow in the third capillary line can be diluted or prepared for the measurement in the second destination detector unit by using suited solvents.
In this context it is preferred that the dilutor pump is connectable to and disconnectable from the third capillary line via second switchable valve means.
This feature enhances the flexibility of the device according to the present invention and allows full automation also in terms of feeding of the solvent into the third capillary line.
In a further preferred embodiment, in the at least one second operating position the third capillary line is connected to at least a second delay line.
The at least one second delay line, which may be chosen with a length different from the first delay line can be advantageously used to store a further peak before feeding same to the second destination detector unit.
In this context it is preferred that in the at least one second operating position of the first valve means the second delay line is connected to the first delay line, which in turn is connected to the third capillary line.
This feature has the advantage that two non-ideally separated chromatography peaks can be stored individually into the two delay lines, from which they can be fed into the second destination detector unit after one another with a suited time delay for a better investigation in the second destination detector unit.
In this context it is preferred that the first valve means is switchable in at least one operating position, in which the second delay line is disconnected from the first delay line.
This feature advantageously improves the possibility to separate two non-ideally separated chromatography peaks. By switching the first valve means into this operating position, the second delay line is disconnected from the first delay line, whereas the third capillary line is connected via the first delay line to the second destination detector unit so that the chromatography peak stored in the first delay line can be fed into the second destination detector unit. After having accomplished this operation, the first valve means can be switched into the at least one second operating position in order to push out the second chromatography peak into the second destination detector unit.
In a further preferred embodiment, the first valve means has at least one further operating position, in which the third capillary line is connected to a drain.
The advantage of this feature is that the chromatography pump can be used to clean parts of or the whole flow path of the device according to the present invention.
In a further preferred embodiment, the first valve means is configured as a turnable multiple port valve, in particular an 8-port valve.
This feature has the advantage that the first valve means is simple in terms of its structure and can be made on a low-cost basis. Further, by virtue of a turnable multiple port valve a simple switching mechanism between the different operating positions mentioned before is achieved.
In a further preferred embodiment, the valve comprises capillaries configured as engravings connecting pairs of the ports of the valve.
This feature has the advantage that the construction of the multiple port valve is further simplified.
Further, according to the present invention, an apparatus for carrying out coupled liquid chromatography (LC) and at least two spectrometry measurements is provided, which comprises a device according to one of the afore-mentioned embodiments.
In a further embodiment of the apparatus, it comprises a flow peak sampling unit for storing single separated peaks of the sample flow.
This feature allows an action to use the second detector unit, in particular in case the second detector unit comprises a MS detector, in the trigger mode to decide if a peak should be stored in a storage loop of the peak sampling unit and to investigate the stored peak later with the first detector unit, e.g. a NMR detector and again with the second detector unit. Further advantages of the peak sampling is the cutting function of cutting the sample flow, thereby increasing the selectivity and the sensitivity of the measurements compared to direct stopped flow measurements.
Such a peak sampling unit is known from U.S. Pat. No. 5,283,036, the disclosure of which is incorporated herein by reference.
In this context the peak-sampling unit can be combined with or replaced by means for concentrating up single peaks stored therein.
This feature has the advantage that the sharpness of single peaks can be increased, i.e. the width of the peaks can be decreased for a better investigation in the first or second detector unit.
In a further preferred embodiment the apparatus comprises control means for automatically or interactive controlling the device of one of the afore-mentioned embodiments.
This feature allows full automatic work of the device and of the entire apparatus as well as a manual interaction with the device.
As already mentioned before, one of the prominent features of the present invention is that the invention renders it possible to use the second destination detector unit, in particular in case that it is a MS detector, also as a decision detector unit for triggering different actions of the total device.
In a further aspect of the present invention, therefore, a method for conducting chromatography combined with spectroscopic measurements on a chromatography flow coming from a flow source in at least a first destination detector unit and in at least a second destination detector unit for spectrometrically investigating single peaks of the chromatography flow is disclosed, wherein at least the second destination detector unit is used as a decision detector unit for selecting peaks of interest, which later are spectrometrically investigated in the second destination detector unit.
By virtue of the method according to the present invention, a totally closed and fully automated chromatography work is possible without any interactions necessary.
In this context, it is useful to use the second destination detector unit as a decision detector for storing single peaks of interest in a peak sampling unit and to use the second destination detector later after transfer of single peaks of interest from loops of the peak sampling unit as an enhanced spectrometer.
In a further preferred embodiment, the first decision detector unit comprises a LC detector, and the first destination detector unit comprises a nuclear magnetic resonance (NMR) detector and the second destination detector unit comprises a mass spectrometer (MS) detector.
As already mentioned before, the present invention is not limited to this LC-NMR-MS system, although the present invention is in particular advantageous for the hyphenation of LC with NMR and MS.
Further features and advantages will be apparent from the following description and the attached drawings.
It will be understood that the above-mentioned features and those to be discussed below, are not only applicable in the given combinations, but may also be employed in other combinations or taken alone without departing from the scope of the present invention.